The present invention relates to methods for releasing an agent under pre-determined conditions, for example at a pre-determined site or in the presence of a pre-determined material, and in particular for releasing an agent for therapeutic, diagnostic or investigative purposes. The invention further relates to pharmaceutical compositions incorporating such methods, materials and kits for use in such methods. It is frequently desirable in the bioscience field to be able to deposit or release a particular agent under, pre-determined conditions, for instance at a specific site within an organism or to mark the presence or absence of an analyte during an assay. At present such specificity is frequently achieved by use of antibodies bound directly to active agents. For instance tumour associated monoclonal antibodies (MABS) have been used to selectively carry chemotherapeutic drugs to tumour cells. Clinical studies have investigated the delivery of methotrexate in patients colorectal carcinoma (Ballantyne et al. 1988. Int. J. Cancer, 42: 103-108) and also the use of adriamycin (see xe2x80x9cPrinciples of Cancer Biotherapyxe2x80x9d Ed. Oldham, R. K., Pub. Raven Press, New York, 1987). Similarly. MABS conjugated to toxins such as ricin, abrin, Pseudomonas toxin, Diptheria toxin and other have also been used as anti-cancer agents. Studies in vitro and in vivo have indicated that such conjugates can be extremely toxic to tumour cells ( Roffler et al. 1991. Cancer Res. 51:4001-4007; Embleton et al 1991; Bri. J. Cancer 63:670-674).
The use of MABS to provide selectivity avoids the side-effect problems associated with traditional chemotherapeutic treatment of cancer either in metastatic disease or in an adjuvant or primary setting. However, a major problem arises because many agents require internalisation before killing the target cell. Additionally immunotoxins usually give rise to unacceptable toxicity due to interaction with non-target cells during passage to the site.
A potential alternative delivery system for selected agents is based around the use of synthetic liposomes. Liposomes were originally described in 1974 (Bangham e al. Methods Membr. Biol. 1: 1-68). Liposomes consist of one or more phospholipid bilayers arranged in concentric rings of alternate aqueous spaces. Many compounds (both lipid and Water soluble) including cancer chemotherapeutics, antimicrobial drugs, enzymes, hormones and nucleic acids have been incorporated into either the aqueous or lipid phase of liposomes. The behaviour of drug-containing liposomes in animal and human subjects has formed the subject of several studies (Gregoriadis 1990, Immunol. Today 11: 89-97).
Thus liposomes offer considerable promise as vehicles for delivery of agents for use in a variety of applications including biochemical and immunological assays, diagnosis, and also pharmaceutical delivery systems for eternal and parenteral use. Unfortunately their application is undermined by the difficulty associated with selectively releasing their contents at a specific time or location.
The present invention has now provided methods for releasing a selected agent at a specific disease site or at a specific time or location and pharmaceutical compositions incorporating such methods, kits and materials for use in such methods, which seek to address some, and in preferred forms all, of the aforementioned problems.
According to a first aspect of the present invention there is provided a method of releasing an agent under predetermined conditions comprising the steps of protecting the agent within a lipid structure, causing lipase activity to be constituted in response to the predetermined conditions, and exposing the lipid structure to the constituted lipase activity such as to release the agent.
By lipase is meant any enzyme which hydrolyses lipids and includes, but is not limited to. enzymes which hydrolyse complex lipids such as phospholipids and grycolipids.
The term constituted as used herein is intended to denote localised, created or significantly increased i.e. a significant achievement or increase in lipase activity is initiated when the predetermined conditions are met.
A large number of naturally occurring lipases are known. For instance many gram-positive and negative bacteria produce enzymes having phospholipase C (PLC) activity. These enzymes hydrolyse phospholipids with varying efficiencies and posses a variety of haemolytic and lethal properties which generally makes them unsuitable for administration to living subjects.
One characterised enzyme is Clostridium perfringens alpha-toxin (CPAT). CPAT promotes direct lysis of certain mammalian cells and is the most toxic PLC described to date (see McDonel, J.L. (1986) pp 617-655 xe2x80x9cPharmacology of Bacterial Toxinsxe2x80x9d Eds. Dorner and Drews, Pub. Pergamon Press, Oxford). CPAT is a peptide containing 370 amino acids.
Preferably the lipid structure employed by the present invention comprises a phospholipid membrane defining a core. More preferably the lipid structure is a liposome. The agent to be released is chosen in accordance with the precise application in which the invention is being employed, however the nature of the agent must be such that it is protectable by a lipid structure.
Preferably the lipase activity employed in the present invention comprises a PLC activity, and more preferably is derived from CPAT. CPAT activity has not previously been demonstrated against liposomes; however the inventors of the present invention have shown that CPAT has significant activity against liposomes.
Preferabiy the lipase activity employed in the present invention is constituted by combining two or more components whereby the lipase activity of the product formed by the components is greater than the sum of the individual components, or alternatively the lipase activity is constituted at a specific location normally with much less or no lipase activity by virtue of the localisation of the lipase either as a holoenzyme or a combination of two or more components in either case in combination with a targeting molecule.
More preferably the components correspond to, or are derived from, an active lipase holoenzyme such that their recombination recovers all or part of the activity of the holoenzyme lipase preferably at a specific location. These components may both be proteinsxe2x80x94however the invention embraces all systems wherein lipase activity is enhanced. localised or recovered by the combination of two or more components, including non-protein components such as co-factors.
Most preferably the components are derived from or include CPAT.
The N-terminal two-thirds of CPAT shares sequence homology with the phosphatidylcholine-PLC from Bacillus cereus. It has been demonstrated that N-terminal recombinant truncated CPAT (aa 1-249) retains phosphatidylcholine hydrolysing activity but has reduced sphingomyelinase activity and is neither haemolytic nor lethal. (Titball et al 1991. Infect. Immun. 59:1872-1874). Recombinant protein comprising the C-terminal third of CPAT (aa 247-370) is devoid of sphingomyelinase and haemolytic activity and is not toxic for murine lymphocytes (Titball et al. 1993, FEMS Microbiology Letters 110: 4550). It has been demonstrated that haemolytic activity (as assessed by an in vitro murine erythrocyte lysis assay) can be restored when the N-terminal and C-terminal recombinant proteins are added together (reconstituted CPAT).
The inventors of the present invention have shown that reconstituted CPAT has significant activity against liposomes.
The pre-determined conditions of the present invention may require that the agent be released only in the vicinity of a tumour or pathogen, or in the presence of an anaiyte or DNA sequence, or under any other suitable detectable condition.
Preferably the achievement of predetermined conditions is causally related to the constitution of lipase activity at a specific location by conjugating at least one of the lipase components or the holoenzyme to a targeting molecule capable of specific binding to a predetermined target under the pre-determined conditions. Suitable targeting molecules include antibodies, antigens, receptors, ligands and nucleic acid probes or primers.
Thus the achievement of conditions may be conveniently related (via a specific antigen-antibody binding event, or the annealing of a nucleotide probe to a specific sequence, or to some other specific physical process) to the constitution of lipase activity in the presence of a predetermined target. Thereafter the addition of suitable lipid structures e.g. liposomes, to the system will effectively lead to target-induced liposomal lysis.
Thus in one embodiment of the invention, which may be used to release an agent at a predetermined target site, the targeting molecule is an antibody which has been raised such as to bind to an antigen on a target site. The antibody is conjugated to a first lipase component such that the component binds at the site. A second lipase component is also added to the system and binds at the site. A second lipase component is also added to the system and binds to the first such that lipase activity is constituted at the site without having had fully active lipase circulating in the system. Liposome containing a suitable agent may be added to the system such that they are lysed on contacting the constituted lipase thereby releasing the agent locally at the site.
It should be noted that the second lipase component may be added independently of in association with, or as an integral part of the liposomes. In another embodiment of the invention which may be used to release an agent at a predetermined target site, the targeting molecule is an antibody which has been raised such as to bind to an antigen on a target site. The antibody is conjugated to a lipase holoenzyme such that the holoenzyme binds at the site. Liposomes containing a suitable agent may be added to the system such that they are lysed on contacting the constituted lipase thereby releasing the agent locally at the site.
Thus these embodiments have in vivo applications for the treatment or locating of a disease through the targeted delivery of membrane-lytic enzyme activity. Unbound anti-body-component conjugate may be cleared from the system prior to adding the second component so as to ensure only local constitution of lipase activity. Specific ablation of the diseased cells or organism may then be achieved by addition of liposomes containing disease modulating compounds. Use of the present invention against tumours may therefore provide an improved killing index due to the ability to release a high local concentration of compound at the tumour and also possible beneficial by-stander effects wherein non-antigen bearing cancer cells in the immediate locality of the tumour will also be the subject of chemotherapeutic killing.
Suitable compounds may include reporter molecules, cytotoxic drugs, lymphokines. anti-inflammatories, anti-fungals, anti-malarials and other drugs combating infectious diseases. synthetic oligonucleotides, nucleic acids (e.g. plasmids) etc, additional antibodies active as immunotoxins, enzymes for conversion of inactive to active pharmaceutical compounds etc. The precise compounds to be used will occur to those skilled in the art according to the problem to be solved.
In a second embodiment of the invention, which may be used for detecting the presence of an antigen in a system, the targeting molecule is an antigen conjugated to a first lipase component. The antigen-1st component conjugate is mixed with antibody raised against authentic antigen such that the antibody binds to the conjugated antigen. The presence of the bound antibody sterically prevents the constitution of lipase activity in the presence of a second lipase component. When the antibody/antigen-component complex is in the presence of authentic antigen the antibody is sequestered by and binds to the authentic antigen. This means that lipase activity will be constituted in the presence of the second lipase component. This event (and hence the presence of authentic antigen) can be detected by addition of liposomes containing a suitable marker e.g. dye, to the system.
Thus this embodiment has in vitro application as an homogenous assay system for the detection of biological or other analytes. The antigen may be conjugated to one of the lipase constituents by recombinant or biochemical techniques.
In another embodiment of the invention, which may be used for detecting the presence of an antigen in a system, the targeting molecule is an antibody conjugated either to a first lipase component or a holoenzyme. The antibody-lipase conjugate may be attached to a specific antigen which has become associated with a solid phase either by direct binding to the solid phase or by binding via an antibody or other intermediate molecule. The presence of antigen can be measured following attachment of the conjugate and washing-off or elution of the excess conjugate by the addition of liposomes containing a suitable marker eg dye.
As an alternative for the detection of an antigen in a system, the antigen could also be attached to a solid phase either directly by absorption or chemical linkage or indirectly via another antibody or binding agent which is, in turn, attached to a solid phase. The antibody-lipase conjugate comprising either the complete lipase or one component is then added followed by if required, the second lipase component and followed by suitable compound-containing liposomes.
In a third embodiment, which may be used for detecting the presence or location of a specific nucleotide sequence in a system, a suitable complementary probe is attached to one or both lipase components such that annealing of the probe or probes to the sequence causes lipase activity to be constituted at that site. This event (and hence the presence of the sequence) can be detected by addition of liposomes containing a suitable marker e.g. dye to the system.
As an alternative for the detection of a specific nucleotide sequence in a system the nucleotide sequence could also be attached to a solid phase either directly by absorption or chemical linkage or indirectly via a complementary nucleotide sequence or an antibody or binding agent which is, in turn, attached to a solid phase. The probe-lipase conjugate comprising either the complete lipase or one component is then added followed by if required, the second lipase component and followed by suitable compound-containing liposomes.
Also embraced by the present invention are materials for use in the methods above.
Thus in a second aspect of the invention there is provided a first lipase component capable of combining with a second lipase component such that the lipase activity of the product formed by the components is greater than the sum of the individual components, said first lipase component being conjugated to targeting molecule capable of specific binding to a predetermined target.
Also embraced by the present invention are pharmaceutical preparations comprising a targeting molecule conjugated with a lipase holoenzyme or lipase component and liposomes containing pharmaceutically active compounds or compounds capable of conversion into pharmaceutically active molecules.
Also embraced by the present invention are kits for use in the methods above.
Thus in a third aspect of the invention there is provided a kit for use in the methods above comprising a first lipase component capable of combining with a second lipase component such that the lipase activity of the product formed by the components is greater than the sum of the individual components, said first lipase component being conjugated to targeting molecule capable of specific binding to a predetermined target, and further comprising the second lipase component.
Preferably the kit still further comprises liposomes containing a suitable agent for use in the methods above.
Preferably the lipase components are CPAT holoenzyme or N-terminal recombinant CPAT and C-terminal recombinant CPAT as herein before described.
A range of alternative lipases may be applicable for use in the current invention. These alternatives include lipases from bovine and porcine pancreas, bee venom, Crotalus venom and include phospholipase B from S.violaceoruber and phospholipase C from Vibrio sp. and B. cereus. It will also be understood that, as an alternative to non-human lipases in pharmaceutical preparations from the current invention, lipases of human origin might be substituted or alternatively non-human lipases such as CPAT might be genetically engineered or modified in order to escape recognition by the human immune system.
Where two or more lipase components are used in the method of the present invention or for substances and materials thereof, it might be possible to improve the strength of association of these lipase components in order to more quickly or more fully reconstitute enzyme activity. This might be achieved by, for example, genetic engineering or by use of auxiliary components which themselves associate thus bringing the lipase components together.
Thus CPAT or reconstituted CPAT activity may be used to direct the lysis of synthetic liposomes containing biologically active preparations or detectable molecule such as dyes thereby providing inter alia a mechanism for targeted drug delivery in vivo, or a reporter system for homogenous and discontinuous assay systems in vitro.
A range of alternative liposomes may be applicable for use in the current inventionxe2x80x94the invention embraces all types of liposome.